The objective of this project is to develop a molecular understanding of amino acid absorption by the small intestinal epithelium. This is accomplished by studying transport events of the separate plasma membranes (brush border and basolateral), and then assimilating the information into a composite picture of transcellular transport. Na-dependent and Na-independent solute transport each play an essential role in gastrointestinal function, yet basic knowledge of transport carriers is incomplete or lacking. This study addresses this problem by focusing on the IMINO contransporter in brush border membranes, and on the "L" carrier in basolaterals. The IMINO system displays an exceptionally selective substrate requirement for L-proline and Na+; the basolateral "L" system is responsibile for 95% of carrier-mediated transport. The project is divided into 3 blocks; within each block, the brush border IMINO transporter and the basolateral "L" transporter are each studied. In each case, proline transport is measured using radioactive tracers and potential-sensitive dye optical techniques. Block 1 concerns the molecular mechanism of carrier transport events. Here, initial flux kinetics will be measured under various conditions of cis & trans substrates, and with imposed membranes PD. Kinetics will be evaluated in light of known mechanistic paradigms, which will provide a comprehensive mathematical model of transport in the terms of an enzymelike reaction mechanism. Block 2 concerns invetigating each transporter's structure/function relationship and membrane-bound in situ functional molecular weight, as probed utilizing high-energy electron radiation inactivation. Block 3 ties together the separate membrane events into a composite molecular description of trans-cellular proline absorption. The proposed project holds long-term health-related implications. This project serves as a prototype, so that the transcellular absorption mechanisms of the other known amino acid carriers can be investigated. This will permit a composite molecular description of amino acid trans-cellular absorption in healthy intestine, will provide a foundation for understanding ion-coupled and ion-independent solute transport, and thus will enhance the understanding of the membrane pathophysiology of genetic defects of amino acid transport in intestinal and renal malabsorption syndromes.